Advances in the field of molecular biology over the last decade have made possible the identification and detailed study of genetically significant regions of specific DNA molecules. A necessary prerequisite to determining the positions of biologically important DNA regions of a given genome was the development of reliable restriction enzyme cleavage mapping techniques. A restriction map defines the sites on specific DNA molecule at which the DNA is cleaved by one or more restriction enzymes. Once a restriction map has been determined for a specific DNA molecule from a particular genome, the resulting map has reference value in any future experimentation involving the same molecule. In addition to providing much useful information about the organization of various DNA molecules, such mapping techniques were also instrumental in the development of DNA sequencing and recombinant DNA techniques.
A number of techniques are currently in use for obtaining DNA restriction maps. The mapping techniques most commonly used generally entail determination of the cleavage fragments in a complete restriction enzyme digest, followed by the tedious ordering of such fragments by various methods, typically involving analysis of a subset of fragments present in each of several overlapping partial enzyme digests. Usually such procedures are time consuming, and may produce less than satisfactory results if a restriction map of high resolution is desired.
One commonly used procedure for obtaining a restriction map is by digesting the DNA molecule of interest, or target DNA, with combinations of restriction enzymes. In order to simplify the procedure, a primary digestion is preferably carried out to limit the number of fragments produced. The primary digest is produced using an enzyme having an infrequent recognition sequence, such as enzymes that recognize hexanucleotide sequences. As is known, the primary DNA fragments may be isolated and recovered following separation, e.g. by electrophoresis. A DNA fragment obtained in the primary digest may be cleaved with additional restriction enzymes, and compared on a gel with appropriate markers of known size. From the data obtained, it is possible to postulate a restriction map which accounts for the observed array of fragments. In building up a restriction map by this procedure, one attempts to assign cleavage sites by trial and error to a uniquely ordered set of locations that are internally consistent with one another.
Resolution of the restriction map obtained by the above-described method of digesting a DNA sequence with a succession of restriction enzymes may be enhanced if the digestion is carried out with different restriction enzymes, both singly and together. By producing fragments from both single and double digestions, a greater array of fragments is generated for use in formulating a restriction map. Similarly, additional specificity concerning the restriction sites in a DNA molecule may be obtained by performing both complete and partial digestions with one or more restriction enzymes.
A variety of computational methods have been developed in an effort to increase the speed and accuracy of the above-described trial and error procedures. One such method uses a computer to calculate the most probable order of restriction sites from single and multiple restriction enzyme digests, Pearson, Nucl. Acids Res., 10: 217-27 (1982). Another algorithm capable of ordering restriction fragments, but requiring only pencil and paper, has also been reported Fitch et al., Gene, 22: 19-29 (1983). Although these approaches are theoretically useful, in practice their application is limited by the requirement for extremely precise length measurements of every fragment in the digest. A missing fragment, even a very small one, or slight inaccuracies in measurement of fragment lengths can produce an erroneous map. In addition, such methods cannot unambiguously order a series of contiguous fragments terminated by the same restriction site.
In preparing a DNA fragment for purposes of restriction mapping, it has been proposed to work from a fixed point on the DNA sequence, for example, from one of the termini of the linear DNA sequence. One such end-labeling method has been reported by Smith and Birnsteil, Nucl. Acids Res., 3: 2387 (1976). According to this method, a DNA fragment is first uniquely labeled at one end of the molecule. The end-labeled fragments are partially digested with a given restriction enzyme. By adjusting the conditions of digestion so that, on the average, only one cleavage occurs per molecule, a ladder of discrete, labeled DNA fragments is generated. The sizes of the resultant fragments reflect the distance between the labeled end of the DNA fragment and a given restriction site. The difference in size between two adjacent fragments on the gel defines the distance between neighboring restriction sites. Although this method yields an accurate restriction map, its general utility is limited, because in order to uniquely end label the-DNA fragments, some prior knowledge of the restriction map is necessary as is the favorable placement of restriction sites.
The mapping technique utilizing end-labeling has been adapted for use in connection with cosmid-type vectors. According to this method, the left or right end of a linear phosmid vector (a cosmid vector derived from the phage .mu.origin of replication) is end-labeled by hybridizing to the vector a labeled oligonulceotide complementary to one end, followed by partial restriction enzyme digestion. This method, however, lacks versatility in that the preparation of phosmid DNA is time comsuming. The method is further limited in its application to cosmid-type vectors.
In view of the currently available procedures for DNA restriction mapping, it would be desirable to provide an improved method which is generally applicable for obtaining unambiguous, high resolution restriction maps of DNA molecules.